T cells obtained from animals immunized against mammary adenocarcinoma 13762 recognize tumor antigens expressed by that cancer. Previously crossreactive antitumor T cells reactive with both adenocarcinoma 13762 and syngeneic ras-transformed Rat1 cells were isolated. Recently the investigator's group has isolated CD4+ T cell clones that recognize shared tumor antigens in vitro and which can eliminate tumor in vivo. Crossreactive T cell clones were demonstrated to be non-reactive with RAS protein. The proposed research shall utilize cloned T-T hybridomas prepared from antitumor T cells reactive with shared tumor antigens to identify tumor antigens by expression cloning of tumor cDNA. One such tumor antigen has already been identified by screening a lgt-11 cDNA expression library prepared from tumor. Deletion mutagenesis has identified a region of the protein which contains the MHC class II binding tumor antigen epitope. The proposed research shall determine the mechanism by which 13762 tumor acquire antigenicity by the following lines of experimentation. cDNAs encoding tumor antigens and also the homologous cDNAs from normal tissue will be isolated and amino acid sequences deduced in order to determine if tumor antigens have acquired amino acid mutations. Expression levels of tumor antigens in tumor and normal tissue will be determined by Northern and immunoblot analysis. In addition, recombinant tumor antigen protein will be prepared as His-tag fusion proteins. Truncation mutants will be created in order to map the MHC binding epitope. Tumor-bearing animals will be immunized with purified recombinant tumor antigen in order to determine if vaccination with a shared tumor antigen that elicits CD4+ T cell immune response is of therapeutic efficacy. The requirement for different host immune cells in vaccination studies will be determined by depletion of selected host immune cell subsets.